eurl-srm - analytical observations report · definition, 4for cf in food of plant origin and honey,...

EU Reference Laboratory for Pesticides Requiring Single Residue Methods

CVUA Stuttgart, Schaflandstr. 3/2, 70736 Fellbach, Germany [email protected]

EURL-SRM - Analytical Observations Report

concerning the following…

o Compound(s): Carbofuran, Benfuracarb, Carbosulfan, Furathiocarb, 3-Hydroxy-Carbofuran o Commodities: Various commodities of plant origin o Method(s): QuEChERS followed by a hydrolysis step o Instrumentation: LC-MS/MS

Analysis of Carbofuran (sum) by applying hydrolysis on QuEChERS extracts Reported by: EURL-SRM

Version 1 (last update: 20.04.2016)

Background information / Initial Observations: Carbofuran (CF) is an insecticide, nematicide and acaricide of the carbamate group. Carbosulfan (CS), furathiocarb (FT) and benfuracarb (BF) are pro-pesticides that degrade to CF, which is the active substance. To account for the high acute neurotoxicity of CF very low toxicological reference values were allocated in 2009 (ARfD 0.00015 mg/kg bw)1 [2]. The metabolite 3-OH-carbofuran (3-OH-CF) was considered exhibiting similar toxicity as the parent. Originally CS, BF and FT were regulated separately from CF and its metabolite 3-OH-CF. The separate MRLs of CS and BF were, however, difficult to enforce as these two compounds are highly susceptible to degradation during extraction and extract storage, especially where the pH of the matrix is low. For this reason, the QuEChERS protocol (EN-15662) foresees not acidifying the extracts after cleanup by dispersive SPE with PSA, to minimize losses between extraction and measurement. But even then, further losses are expected during sample milling, sample extraction (especially in acidic commodities prior to buffering) or storage of homogenates, especially if pH is low and temperatures high. In 2012 the MRLs of CF, BF, CS and FT for most products were lowered to their respective consensus LOQ at that time2. Depending on the commodity groups, the MRLs were set at 0.02*, 0.05* or 0.1* mg/kg for BF and at 0.01*, 0.02* or 0.05* mg/kg for CF, FT and CS. For CS and CF there were also some cases in which CXLs were taken over in EU legislation (e.g. 0.1 mg/kg for CS and 0.5 mg/kg for CF in oranges, mandarins).

A toxicological re-evaluation by EFSA in 20143, however, revealed acute health concerns with these LOQ-based MRLs for CF and with some MRLs of CF, CS and FT. An interim proposal was thus made with MRLs of BF, CS and FT being proposed to be set at the respective consensus LOQs and of CF/3-OH-CF at low levels considering the toxicological reference values established in 2009. The proposals for CF were well below 0.01 mg/kg for food products having high short term consumption (0.001 mg/kg for potatoes, apples and milk and 0.002 mg/kg for

1 Conclusion on pesticide peer review regarding the risk assessment of the active substance carbofuran

(http://www.efsa.europa.eu/en/efsajournal/pub/310r ) , published 10 July 2009 2 Commission Regulation (EU) 2012/899

3 http://www.efsa.europa.eu/sites/default/files/scientific_output/files/main_documents/3559.pdf

mailto:[email protected]

http://www.efsa.europa.eu/en/efsajournal/pub/310r

http://www.efsa.europa.eu/sites/default/files/scientific_output/files/main_documents/3559.pdf



stone fruits, grapes, fruiting vegetables, brassica vegetables, salad plants and stem vegetables). All these MRL*s were calculated to be safe based on the toxicological endpoints of BF, FT and CS (see Table 1).

Table 1: ARfD-Calculations using existing / intermediately proposed MRLs:

Commodity

Carbofuran Carbosulfan Benfuracarb Furathiocarb

MRL * mg/kg

% ARfD

Note MRL * mg/kg

% ARfD

MRL * mg/kg

% ARfD

MRL * mg/kg

% ARfD

Oranges 0.01 60 applying a PF for fruit/pulp of 0.1

0.01 27% 0.02 13% 0.01 22%

Apples 0.001 65% 0.01 20% 0.02 10% 0.01 16%

Tomatoes 0.002 78% 0.01 30% 0.02 15% 0.01 25%

Cucumber 0.002 78% 0.01 30% 0.02 15% 0.01 25%

Potatoes 0.001 103%

slight exceedance acceptable as potatoes are processed

0.01 31% 0.02 15% 0.01 26%

Onions 0.002 106% slight exceedance 0.02 16% 0.05 10% 0.02 13%

Separately regulating BF, CS and FT proved, however, to be of concern not only because of the above-

mentioned analytical difficulties but even more so because of acute risks potentially arising following

degradation of the 3 pro-pesticides to CF during industrial or household processing. Experiments conducted by

the EURL-SRM and shown in this observation report demonstrated that the proposed MRLs for BF, CS and FT

are indeed toxicologically rather critical. Finally it was decided to include CS, BF and FT in the new residue

definition, for CF in food of plant origin and honey, which was established in 20154. For food products having

high short term consumption the MRLs for CF (sum) were set well below 0.01 mg/kg (0.001 mg/kg for

potatoes, apples and milk and 0.002 mg/kg for stone fruits, grapes, fruiting vegetables, brassica vegetables,

salad plants and stem vegetables). For products of animal origin the current residue definition refers to 3-OH-

CF (free and conjugated).

A concern for the laboratories was whether these very low MRLs can be effectively enforced given the 5

components included in the residue definition for CF (sum). A quantitative conversion of CS, BF and FT to CF

during analysis was thus considered highly beneficial, as it reduces the number of compounds to be

analytically determined from five to only two (CF and 3-OH-CF).

4 Commission Regulation (EU) 2015/399




The observations described in the following concern the following topics:

a) Acute risk calculations at different conversion rates of BF, FT and CS to CF b) Conversion rates of BF, FT and CS to CF in a simulated household processing experiment c) Recoveries of BF, CS, and FT using EN-15662 d) Stability experiments of BF, FT and CS in QuEChERS extracts of different pH e) Intentional hydrolysis of BF, FT and CS to CF at various conditions

Compound profile: CF is an insecticide, nematicide and acaricide of the carbamate group. CS, FT and BF are pro-pesticides that

degrade to CF, which is the active substance. None of the four compounds is approved in the EU but there are

still uses in other countries. Carbofuran acts against soil-dwelling and foliar-feeding insects (including

wireworms, white grubs, millipedes, fruit flies, bean seed flies, root flies, flea beetles, weevils, aphids and

thrips) and nematodes in vegetables, ornamentals, beet, maize, sorghum, sunflowers, oilseed rape, potatoes,

alfalfa, peanuts, soya beans, sugar cane, rice, cotton, coffee, cucurbits, tobacco, lavender, citrus, vines,

strawberries, bananas, mushrooms, and other crops. [1] A recent import-tolerance request for mushrooms

from China (exposed through rice straw) was rejected following the toxicological re-evaluation of CF.

Facts at a glance:

Carbofuran

Mode of action Systemic, with predominantly contact and stomach action [1]

LogP 1.8 at 20°C, no effect of pH [2]

Water solubility 315 mg/L at 19.5°C +/- 2.0 °C, no effect of pH [2]

Stability Unstable in alkaline media. Stable in acidic and neutral media.

Decomposes >150 °C [1]

Residue definition EU Food of plant origin and honey: “Carbofuran (sum of carbofuran (including any carbofuran generated from

carbosulfan, benfuracarb or furathiocarb) and 3-OH carbofuran expressed as carbofuran)”

Food of animal origin: “3-OH-carbofuran (free and conjugated) expressed as carbofuran”

Registration Status Not approved within the EU

ADI 0.00015 mg/kg bw/day (2009) [2]

ARfD 0.00015 mg/kg bw (2009) [2]

Main metabolite 3-Hydroxycarbofuran




The following pro-pesticides metabolize to carbofuran (the active component):

Benfuracarb

Mode of action Systemic and contact insecticide with stomach and contact

action [1]

LogP 4.22 (25°C) [2]

Water solubility 8 (20°C, pH 4) [2]

Stability Stable in neutral and weakly basic media, but unstable in

acidic and strongly basic media. Stable up to 190 °C [1]

Residue definition EU See carbofuran


ADI 0.01 mg/kg bw/day (2009) [2]

ARfD 0.02 mg/kg bw (2009) [2]

Furathiocarb

Mode of action Systemic insecticide with contact and stomach action [1]

LogP 4.6 (25°C) [1]

Water solubility 11 (25°C) [1]

Stability Stable up to 400 °C [1]



ADI 0.0035 mg/kg bw/day (RMS 1999) [3]

ARfD 0.006 mg/kg bw (RMS 1999) [3]

Carbosulfan

Mode of action Systemic insecticide with contact and stomach action [1]

LogP 7.42 (25°C, pH-independent) [2]

Water solubility 0.11mg/L (25°C, pH 9) [2]

Stability Hydrolysed in aqueous media [1]


Registration Status Not approved within the EU [3]

ADI 0.005 mg/kg bw/day (2009) [2]

ARfD 0.005 mg/kg bw (2009) [2]




Experiments conducted and observations:

Materials (exemplary5): Carbofuran (purity 99,5%), purchased from Dr. Ehrenstorfer (Cat #: C11010000) Benfuracarb (purity 92%), purchased from Dr. Ehrenstorfer (Cat #: CA10475000) Furathiocarb (purity 99%), purchased from Dr. Ehrenstorfer (Cat #: C13970000) Carbosulfan (purity 98%), purchased from Dr. Ehrenstorfer (Cat #: CA11030000) 3-Hydroxycarbofuran (purity 98%), purchased from Dr. Ehrenstorfer (Cat #: C 11011000)

Table 2: Instrumentation details: LC WATERS Acquity UPLC

MS/MS ABSCIEX 5500 Q-Trap, run in ESI positive mode

MRMs 222.2/122.9; 222.2/165.1

Column Acquity BEH C18, 2.1x100 mm, 1.7 µm

Pre-column Acquity BEH C18, 2.1x5 mm, 1.7 µm

Mobile Phase A: 5 mmol NH4formate in purified water + 5% Methanol B: 5 mmol NH4formate in Methanol

Gradient Time (min) Mobile Phase A (%) Mobile Phase B (%)

0 100 0

0.5 60 40

7.5 10 90

11.0 10 90

11.1 100 0

14.0 100 0

Flow 0.4 mL min-1

Injection volume 2 µL, partial loop with needle overfill

Column temperature 40°C

Table 3: MS/MS parameters (exemplary ABSciex 5500 Qtrap) Substance Intensity Q 1 Q 3 DP CE CXP

Carbofuran 1

222 123 76 29 6

2 165 76 17 14

Benfuracarb

1

411

190 71 17 10

2 102 71 45 6

3 74 71 67 2

Furathiocarb

1

383

195 96 27 10

2 252 96 19 2

3 167 96 35 8

Carbosulfan

1

381

118 86 29 20

2 160 81 21 14

3 76 76 53 6

3-Hydroxy-carbofuran

1

238

163 76 21 10

2 181 76 17 10

3 220 76 11 40

5 Disclaimer: Names of companies are given for the convenience of the reader and do not indicate any

preference by the EURL-SRM towards these companies and their products




a) Acute risk calculations at different conversion rates of BF, FT and CS to CF:

To start with, a theoretical exercise was conducted to demonstrate that already small conversion rates of BF,

CS and FT to CF during food processing can potentially lead to toxicologically critical CF levels, even if the

proposed MRLs for the three pro-pesticides were not exceeded.

The theoretical exercise was based on commodities with high portion sizes, namely oranges, cucumbers,

tomatoes, apples, potatoes and onions. For these products the proposed MRLs were 0.01 mg/kg for CS and FT

and 0.02 mg/kg for BF. Assuming residue levels at the supposedly safe MRLs proposed and considering the

molecular weight factors between CF and the three pro-pesticides, it was calculated to which percentage the

IESTI for CF was covered/exceeded if the conversion rates to CF were 10%, 20%, 30%, 50% or 100%. This

calculation was done for each pro-pesticide commodity combination. Variability factors and the peeling factor

in the case of oranges were already considered in the IESTI calculations done by EFSA.

These calculations are shown in Table 4a and 4b. Assuming a 0.01 mg/kg level (the intermediately proposed

MRL for FT and CS in these food products) already a conversion rate of 20% of any of the three pro-pesticides

to CF would theoretically lead to an ARfD exceedances of CF in the case of potatoes and onions. The same

applies for apples at a conversion rate of 30% and for tomatoes and cucumbers at a conversion rate of 50%.

Assuming a 0.02 mg/kg level (the intermediately proposed MRL for BF) already a conversion rate of 10% of

any of the three pro-pesticides to CF would theoretically lead to an ARfD exceedances of CF in the case of

potatoes and onions. The same applies for apples at a conversion rate of 20% and for tomatoes and

cucumbers at a conversion rate of 30%.

To account molecular weight differences the following conversion factors:

Conversion pair Factor

CF to BF 1.855

CF to FT 1.73

CF to CS 1.72




Table 4: Theoretical calculation of Acute Risks associated with the conversion of CS, BF or FT to CF during

processing assuming different conversion rates. Pesticide/ Commodity/ Conversion rate – combinations

where the IESTI (100% ARfD coverage) is exceeded are highlighted in red.

a) Assuming the initial level of CS, BF or FT as being 0.01 mg/kg

b) Assuming the initial level of CS, BF or FT as being 0.02 mg/kg




b) Conversion rates of BF, FT and CS to CF during simulated household processing

A simple experiment was conducted to get a first idea of the behavior and conversion rates of CS, BF

and FT to CF in processed food items. Two different scenarios were chosen:

i. Finely sliced cucumbers left standing for 4h at RT

ii. Grated apple, slightly sprinkled with lemon juice and left standing for 4h at RT

CF, BF and FT were separately spiked at levels representing their proposed MRLs in apples and

cucumbers and the CF generated was determined using a procedure that minimizes degradation of

CS, BF and FT (QuEChERS with no acidification following dSPE cleanup with PSA sorbent). The

conversion rates observed for each compound are shown in Table 4. For BF and CS the CF levels

generated within the 4 hours storage clearly exceeded the toxicologically critical threshold. Same

applies for CS in the case of apple. Interestingly, despite the higher pH, conversion in cucumbers was

more pronounced than in apples. Also conversion rates of FT were higher than of BF and CS, which is

in contrast to the observations made during extraction and extract storage, where FT was

significantly more stable than BF and CS. This suggests an enzymatically assisted decomposition.

Carbosulfan

(CS)

Benfuracarb

(BF)

Furathiocarb

(FT)

MRLs

CF

100%

ARfD

(IESTI)

Experimentally

determined

conversion

rate to CF

10 µg/kg*

CS will

covert to

x µg/kg CF

Experimentally

determined

conversion

rate to CF

20 µg/kg*

BF will

covert to

x µg/kg CF

Experimentally

determined

conversion

rate to CF

10 µg/kg*

FT will

covert to

x µg/kg CF

µg/kg

(ppb)

µg/kg

(ppb)

Apple 33% 1.94 27% 2.88 48% 2.75 1 1.54

Cuccumber 5% 0.27 43% 4.62 76% 4.37 2 2.56

*Concentrations corresponding to intermediately proposed MRL

Table 5: Observed conversion rates of CS, BF and FT to CF in two different household processing scenarios; a) Finely sliced cucumbers left standing for 4h at RT; and b) Grated apple, slightly acidified with lemon and left standing for 4h at RT

and calculation of the acute risks associated with the released CF in case CS, BF and FT were present at levels corresponding to their MRLs




c) Recoveries of BF, CS, and FT using EN-15662

Separate recovery experiments of BF, CS and FT in cucumber and orange juice were conducted using the

QuEChERS method (EN 15662) with a 15 min first extraction step. The commodities were spiked at 0.1 mg/kg

using standard solutions of FT, CS and BF in pure acetonitrile. These were shown to contain some CF as

follows: The FT solution contained 0.2% CF (expressed as FT); the CS solution 0.9% CF (expressed as CS); and

the BF solution 5% CF (expressed as BF). For the sake of simplicity the deviations in the initial concentration of

the parents and CF were disregarded in the following calculations.

The QuEChERS extracts were analyzed either directly or following dSPE with MgSO4/PSA without acidification

(as proposed in EN15662).

i. Analysis from raw QuEChERS extracts

By the time of the measurement of the raw orange juice extracts (4h after extraction) BF parent practically

disappeared and CS dropped by >90%. By the time of the measurement of the raw cucumber extracts (2h after

extraction), BF parent dropped by 85% within 2h and by 95% within 3h whereas CS dropped by 44% within 2h

and by 60% within 3h. FT was quite stable with CF corresponding to ca 0.6 % FT being formed in raw cucumber

extracts within 3-4 h and to ca. 1% in raw orange juice extracts within 6h.

The decline of BF and CS concentration was, however, not accompanied by a proportional increase of CF.

Apparently due to the formation of intermediate products the formation of CF was considerably delayed.

Interestingly, and although the degradation rates in orange juice extracts were higher than in cucumber

extracts the formation rate of CF in cucumber was less delayed than in orange juice extracts. As a result the

summed recovery of BF / CS plus CF in the case of orange juice extracts was much lower (ca. 30% for BF and

ca. 40% for CS) compared to cucumber extracts (ca. 90% for BF and ca. 95% for CS).

Similar observations were made when injecting standard solutions of BF and CS based on blank raw extracts of

orange juice and cucumber. In the solutions based on orange-juice BF losses within 3 h after preparation were

already >99% with only 30% of the original BF being converted to CF. CS losses were 83% with only 28% being

converted to CF. In the solutions based on cucumber raw extract BF losses within 3 h were as high as 71% with

50% of the original BF being converted to CF and CS losses were 39% with 26% being converted to CF.

Overall these results indicate that measurement of BF and CS from raw extracts is critical. Losses occur both

during extraction and storage of the extracts in vials with the latter factor contributing more, especially if

measurement is delayed.




Table 6: Recovery experiments for BF, CS, FT with analysis of CF, BF, CS and FT

in QuEChERS RAW Extracts (Calibration in pure acetonitrile)

Spiked at

0.1 mg/kg

to the matrix

#

Time

elapsed (between

extraction and

measurement)

Cucumber

Time

elapsed (between

extraction and

measurement

Orange juice

%

parent

left

% CF formed

(expr. as

parent)

Sum

%

parent

left

% CF formed

(expr. as

parent)

Sum

BENFURACARB

(BF)

1 2 h 15 70 85

4 h 0.1 30 30

2 2.5 h 14 73 87

5 h 0.0 30 30

3 3 h 5 87 93

5.5 h 0.0 33 33

ø 11 77 88 0.03 31 31

CARBOSULFAN

(CS)

1 2 h 56 34 90

4 h 9 33 42

2 2.5 h 43 53 96

5 h 6 35 41

3 3 h 40 59 99

5.5 h 5 36 40

ø 46 49 95 7 35 41

FURATHIOCARB

(FT)

1 2.5 h 95 0.5 96

4.5 h 104 0.9 105

2 3 h 96 0.6 99

5.5 h 101 1 102

3 3.5 h 99 0.6 100

6 h 103 0.9 104

ø 98 0.6 98 103 0.9 104

ii. Analysis from QuEChERS extracts following dSPE with MgSO4 /PSA without acidification

The extracts of the recovery experiment described above were thus additionally injected following dSPE

cleanup (with MgSO4 /PSA) and no acidification afterwards (in the following abbreviated as CNA extracts =

Cleaned-up but Not Acidified). This procedure is recommended in the EN 15662 (QuEChERS standard) for CS,

BF and FT (at that time these compounds were separately regulated). Cleanup with PSA causes the pH to rise

and reduces degradation. The pH of the extracts in the case of cucumbers was 7.6.

To check the stability of BF, CS and FT during the storage of CNA extracts (following dSPE with PSA, without

acidification) standard solutions based on blank CNA-extracts orange juice and cucumber extracts were

prepared. In the case of cucumbers CNA extracts spiked with CS, the levels of CF increased from ca 1% (level in

the acetonitrile working standard) to ca. 3% (expressed as CS) within 8h. In extracts spiked with BF the CF

levels increased from 5% to ca 11 % (expressed as BF). In the case of orange juice CNA extracts spiked with CS,

the levels of CF increased from ca 1% to ca. 2% (expressed as BF) within 11h. In extracts spiked with BF the CF

levels increased from 5% to ca 8 % (expressed as BF). In all cases losses of the parents were minor.




This indicates that following cleanup with PSA degradation of BF and CS slows down considerably. This

measure will however not compensate the losses during the extraction procedure.

Analysis of CNA-extracts of the recovery studies showed considerably higher losses (see Table 7), indicating

that they mostly occurred prior to cleanup with PSA. In the case of cucumbers losses of BF ranged between 25-

30%, with the CF levels rising to ca. 30% (expressed as BF). Deducting losses of ca. 5% which occurred during

the storage of CNA-extracts (over 9-10 hours) the BF losses actually occurring during the extraction step were

expectedly in the range of ca. 20-25%. CS losses under the same conditions were in the range of 15 %

increasing to ca 25%. In the case of orange juice BF losses were in the range of 45-55% with most of them

occurring during the extraction step. The CF formed was in the range of 35% (expressed as BF). CS losses

ranged around 25% with ca. 20% CF being formed (expressed as BF). CS losses under the same conditions were

in the range of 15 % (expressed as CS). Employing the orange juice samples for extraction at room

temperature, BF losses increased further by ca. 10% as CS-losses by ca. 7%.

Table 7: Recovery experiments for BF, CS, FT with analysis of CF, BF, CS and FT

in QuEChERS Extracts following dSPE-cleanup with PSA and no acidification

(Calibration in pure acetonitrile)

Spiked at

0.1 mg/kg

to the matrix

#

Time

elapsed (between

extraction and

measurement)

Cucumber

Time

elapsed (between

extraction and

measurement)

Orange juice

%

parent

left

% CF formed

(expr. as

parent)

Sum

%

parent

left

% CF formed

(expr. as

parent)

Sum

BENFURACARB

(BF)

1 9 h 75 27 102

11.5 h 46 36 82

2 9.5 h 69 28 98

12.5 h 53 36 89

3 10 h 76 34 110

13 h 48 37 85

ø 73 30 103 49 36 85

CARBOSULFAN

(CS)

1 9 h 91 10 100

11.5 h 74 20 94

2 9.5 h 85 13 97

12.5 h 73 20 93

3 10 h 82 12 95

13 h 72 19 91

ø 86 12 97 73 20 93

FURATHIOCARB

(FT)

1 9,5 h 100 0.5 101 12 95 1.2 96

2 10 h 101 0.6 102 13 94 1.2 95

3 10.5 h 104 0.5 105 13.5 97 1.3 98

ø 102 0.5 103 101 1.2 102

Overall these results indicate that the analysis of BF and CS using QuEChERS is critical especially in the case of

acidic samples. When analyzing raw extracts the main part of the losses occurs during the waiting time

between extraction and measurement. Under acidic conditions the formation of CF is delayed leading to

reduced summed recoveries. In a routine analysis (e.g. for screening) this could lead to underestimations or




even false negative results. When CNA-extracts are measured losses occurring during the extraction step play

the main role. Recovery rates of the parents are acceptable and the conversion rates of BF and CS are

increased. Thus, in theory, summed analysis of the CS or BF plus CF is possible. Given the very low MRLs

conversion of the parents to CF is however preferred. FT proved to be very stable in both raw extracts

experiencing practically no losses.

d) Conversion of BF, FT and CS to CF in QuEChERS extracts of different pH

In order to study how CS, BF and FT behave in extracts having different pH, different types of extracts were

prepared as follows, spiked and analyzed by LC-MS/MS after certain time intervals.

a) Raw extracts (pH ̴ 4 for cucumbers and pH ̴ 3.6 for oranges) b) Raw extracts slightly acidified with 50µL of 5% formic acid in ACN (pH ̴3.2 for cucumbers) c) Raw extracts strongly acidified with 10µL of 99% formic acid (pH ̴2.56 for cucumbers and oranges) d) Extracts after cleanup with PSA but not acidified (“CNA extract”; pH 7.6 for cucumbers) e) “CNA extracts” acidified 10µL of 5% formic acid (10%) (pH ̴ 4 for cucumber and ca. 4.4 for orange)

f) “CNA extracts” after adding 10µL of 25% ammonia (pH ̴ 9.7 for cucumber and ca. 9.4 for orange) g) Raw extracts acidified with 10 µL H2SO4

a) Raw QuEChERS extracts (pH: ̴4 for cucumbers and pH: ̴3.6 for oranges)

Raw QuEChERS extracts of cucumbers (pH ̴4.0) and oranges (pH ̴3.6) were separately spiked with BF, CS or FT

and left standing at room temperature determining at certain time intervals a) the residual parent compounds;

and b) the conversion rate to CF. After ensuring that matrix effects do not affect measurement, calibration

solutions for CF, BF, FT and CS in pure acetonitrile were used. These were prepared by appropriately diluting

the spiking solutions and were found to contain either non-measurable or very insignificant levels of CF.

Already within the few minutes (0h) after spiking the extracts, CS losses were in the order of 20%. After 2h CS

and BF dropped by 67% and 40% respectively in cucumber extract and by 90% and 52% respectively in the

more acidic orange extract. After 18h no BF was left in any of the extracts whereas CS dropped to 8% in

cucumber extract and 1% in orange extracts. As in the previous experiment the degradation rate did not

correlate with the formation of CF, indicating that certain intermediate products are formed. After 18h,

conversion yields of BF to CF in the cucumber extract were only 50% rising to 83% after 6 days. In orange

extracts the conversion rates of BF to CF were 37% after 18h and 61% after 6 days. Similar observations were

made for CS with conversion rates to CF after 6 days reaching 85% in cucumber and 64% in orange extracts. FT

proved to be resistant to hydrolysis in raw QuEChERS extracts, with very minor losses being noticed even after

6 days of extract storage.

6 pH values as measured with a pH electrode




b) Raw extracts acidified with 10 µL 99% formic acid (pH 2̴.5 for cucumbers and oranges)

The strongly acidified raw QuEChERS extracts were separately spiked with BF and CS and measured after 2

hours. In both cucumber and orange extracts BF and CS disappeared after two hours. Conversion rate of BF

and CS to CF was quantitative in cucumber but only ca. 60% in oranges for both CS and BF.

c) Raw extracts acidified with 50 µL of 5% formic acid in ACN (pH 3̴.2 for cucumbers)

The slightly acidified raw QuEChERS extracts were separately spiked with BF, CS and FT and measured after 6

hours and 5 days. FT remained stable. In both cucumber and orange extracts BF and CS disappeared after 6

hours. After 6 hours the conversion rate to CF was ca 90% in the case of BF and ca. 85% in the case of CS. After

5 days both the conversion rate was quantitative in both cases.

d) Extracts after cleanup with PSA but not acidified (“CNA extract”; pH ̴7.6 for cucumbers)

The CNA extracts were separately spiked with BF, CS and FT and measured after 5d. All compounds were stable.

e) “CNA extracts” acidified 10 µL of 5% formic acid (10%) (pH ̴ 4 for cucumber and ̴ 4.4 for orange)

All three compounds were spiked (FT, BF and CS) and measured after 2 hours, 6 hours and 18 hours. FT remained stable. In cucumber BF dropped by ca 90% after 18 hours with the CF formed reaching only ca. 45% (expressed as BF). CS dropped by ca. 55% after 18 hours with the CF formed reaching only ca. 28% (expressed as CS). In oranges BF dropped by ca 80% after 18 hours with the CF formed reaching only ca. 14% (expressed as BF). CS dropped by ca. 40% after 18 hours with the CF formed reaching only ca. 9% (expressed as CS).




f) “CNA extracts” after adding 10 µL of 25% ammoniac (pH ̴ 9.7 for cucumber and ca. 9.4 for orange)

Only FT was spiked and remained stable over 2 days. In conclusion Carbosulfan and Benfuracarb tend to rapidly degrade in acidic extracts with CF being formed with some delay (formation of intermediate products). Furathiocarb is quite stable at acidic as well as at alkaline conditions at room temperature.

g) Raw extracts acidified with 10 µL 5N H2SO4 and heated up

Experiments b) and c) where acidification with formic acid was employed showed that hydrolysis of BF and CF

to FT was possible with the formation of CF increasing with time and eventually becoming quantitative after

some hours. FT, however proved to be much more resistant to acidic hydrolysis. It was thus decided applying

elevated temperature during the acidic conversion in the QuEChERS extract.

When extending hydrolysis time to 6h no significant drop of CF was noticed indicating the CF is quite stable

under these hydrolysis conditions.

Hydrolysis Time at 80°C

Conversion rate to CF (expressed as BF or CS or FT)

BF CS FT

2h 104 96 92

4h 103 96 95

6h 104 102 95

Carbofuran phenol was shown not be formed at any significant levels. The use of Chlorpyrifos-D10 as internal

standard is critical as it degrades under these hydrolysis conditions. The formation of 3,5,6-tricloropyridinol

(TCPy) was observed. Carbofuran D3 is better suitable as IS.




The final method developed is as follows:

Extraction: Apply the citrate buffered QuEChERS (EN 15662). Weigh 10 g of frozen fruit or vegetable

homogenate or 5 g of cereals; adjust water content to 10 mL where necessary, add 10 mL acetonitrile and

internal standard (e.g. 100 µL of an appropriately concentrated solution of Carbofuran-D37). Shake 15 min

using a mechanical shaker. Add a mixture of 4g MgSO4, 1g NaCl, 1 g trisodium citrate dihydrate and 0.5 g

disodium hydrogen citrate sesquihydrate, shake 1 min and centrifuge.

Cleanup: Cleanup via dispersive SPE is optional for fruits and vegetables.

Hydrolysis: Transfer 1 mL of raw extract into vial and add 10 µL 5N H2SO4. Nearly quantitative transformation

of BF, CS and FT into CF is achieved by heating the vials for 3h at 80°C.

LC-MS/MS analysis: For screening purposes CF, 3-OH-CF as well as BF, FT and CS may be analyzed by LC-

MS/MS directly in QuEChERS raw extracts or cleaned-up extracts. In case of positive findings the hydrolysis

step can be conducted as described above and LC-MS/MS analysis of CF repeated.

1mL final extract will represent approximately 1 g matrix.

For measurement conditions and recovery figures see EURL-SRM - Analytical Method Report (Analysis of

Residues of Carbofuran (sum) using QuEChERS method).

References

[1] e-Pesticide Manual V5.0, Author: C. D. S. Tomlin; 15th Edition, Version 5.0, 2009

Publisher/Contact: The British Crop Protection Council

[2] EFSA

[3] EU Pesticides Database

Document History

Date Action Changes

Apr. 2016 Publication of V1

7 Before using an internal standard, check if it is stable under the hydrolysis conditions


eurl-srm - analytical observations report · definition, 4for cf in food of plant origin and honey,...

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